The present invention relates to a method and an apparatus for reacting solid and liquid phases. More specifically, it relates to a method of efficiently reacting a reactive substance bonded on a solid phase and a reactive substance in a liquid phase, and an apparatus used for carrying out the method.
There has been known an immunological method which employs an antigen-antibody reaction for determining the quantity of a very small amount of substance in body fluids, or for determining the concentration of an administered medicine in blood or urine in an organism. Several methods, which are based on different principles of determination, have been known and put in practical use. They include radio-immunoassay (RIA), enzyme-immunoassay (EIA) and fluorescent-immunoassay (FIA), which have been widely employed because of their high sensitivity, and their high effectiveness in quantitative determination. When these assays are carried out, the so-called sandwich method or the competitive method is employed as an assay principle. Particularly sandwich method is widely used, since it provides a high degree of analytical sensitivity and is easy to carry out.
According to the sandwich method, the antigen to be measured is reacted with an insolubilized corresponding antibody (first reaction), whereby an antigen-antibody complex is formed. This complex is reacted with an antibody labeled with a labeling agent and capable of combining with the antigen to be measured (labeled antibody) (second reaction). Then, the labeled antibody is divided into two portions; one has combined to the antigen-antibody complex, and another has not, and the activity of the labeling agent in either portion is measured. On the other hand, similar procedures are repeated for an antigen at known concentrations to establish a calibration curve. The quantity of the antigen to be determined is obtained from the calibration curve. The labeling agent may be, for example, an enzyme, or a radioactive or fluorescent substance.
According to the competitive method, which has been first employed in the radio-immunoassay, the measurement is carried out as follows:
When the antigen to be measured and a given amount of the labelled antigen are reacted with the insolubilized antibody corresponding to the antigen to be measured, both antigens competitively combine with the insolubilized antibody. Next, the labelled antigen is divided into two portions; one has combined to the insolubilized antibody and another has not, and the activity of the labelling agent in either portion is measured. On the other hand, similar procedures are repeated for an antigen at known concentrations to establish a calibration curve. The quantity of the antigen to be determined is obtained from the calibration curve.
In carrying out these reactions, it is advantageous to use the inner wall surface of a vessel as a carrier for a reactive substance such as an antibody to be insolubilized. For example, a plastic test tube is often used, since it serves both as a carrier for insolubilization and as a reaction vessel and it is easy to handle. The inner wall surface of a reaction vessel is, however, disadvantageous in that its surface area to which an antibody or other reactive substance is fixed is smaller than in the case of other carriers, such as plastic beads, filter paper or cellulose particles, and therefore can carry only a smaller quantity of the reactive substance. Consequently, a reaction time therefore becomes longer according to the conventional method in which a test tube or other reaction vessel carrying an insolubilized reactive substance on its inner wall surface is kept upright to stand still, or its contents are stirred intermittently.